Dear community,

I am working with hiPSC derived pancreatic cell clusters. I am using the Protocol from Rezania et al. 2012 for generation of hormone expressing cell clusters as well as the air lift culture protocol (Rezania et al. 2014).

Immunofluorescent staining of the generated cells gave me nice signals for Glucagon and Somatostatin, but poor staining Insulin+ beta-cells. As alpha and delta cells are detectable, I think the problem is the Insulin staining rather than the protocol. The insulin antibody was tested on other samples and worked fine. I read that insulin epitops are quite prone to get lost during fxation. So, I think there might be a mistake in sample prepartion (overfixation etc.).

I prepared my cells as following for staining:

- 2 h 4 %PFA at RT

- 1x 10 min wash with PBS-

- Paraffin embedding

- Descending EtOH

- Antigen retrieval: 20 min, 100°C, Citrate Buffer pH 6.0

- 5 min PBS- + 0.5% Tween

- Blocking: 5% BSA

- Primary antibody overnight

Does anybody have a protocol for fixation and paraffin embedding of hiPSC derived pancreatic endocrine cells for subsequent staining of insulin? Do you have other suggestions how to improve quality of the sample and the staining?